Turnover of Proteins as a Controller of Soil Nitrogen Cycling

自然和管理生态系统中的植物生产力往往受到氮(N)的限制，植物从土壤中获得的氮主要以铵或硝酸盐的形式存在--这两种形式都是N的无机形式。然而，土壤中95%以上的N以有机形式存在，主要是来自腐烂的植物材料或分解植物材料的微生物的酶和其他蛋白质。这项研究将研究森林土壤中蛋白质的分解，这是将有机氮转化为植物可用形态的主要瓶颈。蛋白质被细菌和真菌产生并释放到土壤环境中的酶(蛋白水解酶)分解。蛋白质与土壤-矿物质和有机成分之间的相互作用可以影响微生物蛋白酶的活性。这项研究将直接测量微生物和土壤对蛋白质降解的控制，从而更深入地了解植物和其他环境中氮的供应瓶颈。这项研究具有变革的潜力，因为它将揭示耦合碳(C)和氮循环的核心机制。需要了解蛋白酶如何解聚和循环有机氮，以更准确地模拟有限的自然氮供应如何影响C&N循环之间的相互作用。研究将通过实验室以外的四个活动传达给科学中代表性较低的群体：(1)安德鲁斯长期生态研究(LTER)站点的年度HJA日，接触到大量公众；(2)协助每年夏天为初中和高中教师教授的土壤科学短期课程；(3)为K-12教师提供研究经验；以及(4)为研究生设计环境蛋白质组学课程和教授短期课程。有机N周转一直被认为是土壤生态系统N有效性的关键控制因素，但将大分子N化合物分解为可同化N单体的过程很少受到关注。这项拟议的研究将研究有机氮的周转，特别是构成土壤有机氮的最大部分的蛋白质。提出了一种新的土壤氮循环的概念方法，该方法认识到有机氮的生物有效性受到“基质”和“微生物”的控制：与土壤基质相关的相互作用的化学和物理过程控制蛋白质氮对复杂微生物群落产生的各种酶的可获得性，以响应环境刺激。该模型将通过两个目标进行探索：(1)确定两种具有不同物理化学特征的15N标记的‘底物’蛋白质在土壤中的去向，跨越资源可获得性、矿物学和微生物生态的梯度，特别强调蛋白质-矿物质和蛋白质-有机物相互作用作为基质调节因素的相对重要性；(2)确定不同微生物群对土壤中不同N有效性的蛋白酶活性的相对贡献，以及通过C-N限制对蛋白酶活性的控制，以探索蛋白质周转的微生物调节。与这些目标相关的研究问题将使用温带森林中特征良好的长期实验的土壤来解决。15N标记的蛋白质将被用来确定它们的周转率以及蛋白质中C和N的去向。这将伴随着对蛋白酶的表征和对其活性的测量。将进行操纵性实验，以确定细菌和真菌对蛋白酶活性的相对贡献。这些数据将为我们在有机氮周转的概念模型中发挥作用的控制机制提供洞察。总而言之，目标1和目标2中产生的数据将使我们能够确定土壤基质和微生物群落在蛋白质N在土壤中降解时对其命运所起的机械作用。它将揭示微生物蛋白酶在土壤生态系统中的活性是如何变化的，并通过它们的催化类型来表征它们。了解蛋白酶的性质和行为对于协调它们如何获得不同形式的土壤相关蛋白质至关重要，因此是成功进行未来研究的基本前提。为此，拟议的研究将产生土壤中氮素转化和保持的定量信息，这对土壤生产力和环境健康的管理也是至关重要的。拟议的研究产生的数据和功能关系最终打算与N循环模型相结合，该模型分离微生物功能群或酶的活动。这项研究的结果将包括更定量地了解微生物蛋白酶在陆地生态系统有机氮循环中的作用，以及蛋白酶的来源和多样性。

Plant productivity in natural and managed ecosystems is often limited by nitrogen (N), which plants obtain from the soil mainly as ammonium or nitrate - both of which are inorganic forms of N. Yet, more than 95% of the N in soil exists in organic forms, primarily enzymes and other proteins that originated from decaying plant material or from the microorganisms that decompose it. This research will study protein breakdown in forest soil, which is the major bottleneck in converting organic N into forms available to plants. Proteins are broken down by enzymes (proteases) that are produced and released into the soil environment by bacteria and fungi. Interactions between proteins and soil-mineral and organic components can affect the activity of microbial proteases. This research will directly measure microbial and soil controls on protein degradation and thus provide a deeper understanding of the bottleneck in the supply of N to plants and other environmental fates of N. This research has the potential to be transformative in that it will reveal a core mechanism coupling carbon (C) and N cycling. Understanding how proteases de-polymerize and recycle organic N is needed to more accurately model how limited natural nitrogen availability affects interactions between C & N cycles. Research will be conveyed to groups underrepresented in science through four activities that extend beyond the laboratory: (1) the annual HJA Day at the Andrews Long-Term Ecological Research (LTER) site, which reaches large numbers of the general public; (2) assist in a short-course in soil science that is taught each summer for middle and high school teachers; (3) provide a K-12 teacher with research experience; and (4) design curriculum and teach a short-course in environmental proteomics for graduate students.Organic N turnover has long been recognized as key controller of N availability in soil ecosystems, but the processes that break down macromolecular N compounds into assimilable N-monomers have received little attention. The proposed research will examine the turnover of organic N, particularly proteins, which make up the greatest fraction of soil organic N. A new conceptual approach towards the soil N cycle is proposed that recognizes "matrix" and "microbial" controls on the bioavailability of organic N: Interacting chemical and physical processes associated with the soil matrix control the accessibility of proteinaceous N to a diverse complement of proteases produced by a complex microbial community in response to environmental stimuli. This model will be explored through two objectives: (1) determining the fate of two 15N-labeled 'substrate' proteins with distinct physicochemical characteristics in soils across a gradient of resource availability, mineralogy, and microbial ecology, with special emphasis on the relative importance of protein-mineral and protein-organic matter interactions as contributors to matrix regulation; (2) determining the relative contributions of different microbial groups to protease activity in soils that vary in N availability, and the control of protease activity by C versus N limitation as a means of exploring the microbial regulation of protein turnover. Research questions associated with these objectives will be addressed using soils of well-characterized, long-standing experiments in temperate forests. 15N-labeled proteins will be used to determine their turnover rates and the fate of the C and N in the proteins. This will be coupled with characterization of proteases and measurements of their activities. A manipulative experiment will be done to determine the relative contribution to protease activity by bacteria and fungi. These data will provide insight into the control mechanisms functioning in our conceptual model of organic N turnover. Collectively, data generated in Objectives 1 and 2 will allow us to identify the mechanistic functions that the soil matrix and microbial community exert on the fate of protein N as it is degraded in soils. It will reveal how the activities of microbial proteases vary among soil ecosystems and characterize them by their catalytic types. Knowing the properties and behavior of proteases is essential to reconcile how they access the different forms of soil-associated proteins, and thus constitutes a fundamental prerequisite for successful future studies. To this end, the proposed research will generate quantitative information of N transformation and retention in soil, which is also critical to the management of soil productivity and environmental health. The data and functional relationships generated by the proposed research are eventually intended to be coupled to N cycling models that separate the activity of microbial functional groups or enzymes. The outcomes of this research will include a more quantitative understanding of the role of microbial proteases in organic N cycling in terrestrial ecosystems and of the sources and diversity of proteases.